Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli

BACKGROUND: Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H(2) production involves consumption of 2H(+), hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2-2.5) that are three pH units lower than the pH limit of growth (pH 5-6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms. METHODS AND PRINCIPAL FINDINGS: We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H(2) to 2H(+). Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H(2) production and consumption was tested using a H(2)-specific Clark-type electrode. Hyd-3-dependent H(2) production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H(2) consumption was maximal at alkaline pH. H(2) production, was unaffected by a shift in external or internal pH. H(2) production was associated with hycE expression levels as a function of external pH. CONCLUSIONS: Anaerobic growing cultures of E. coli generate H(2) via Hyd-3 at low external pH, and consume H(2) via Hyd-2 at high external pH. Hyd-3 proton conversion to H(2) is required for acid resistance in anaerobic cultures of E. coli

A Requirement of TolC and MDR Efflux Pumps for Acid Adaptation and GadAB Induction in Escherichia coli

BACKGROUND: The TolC outer membrane channel is a key component of several multidrug resistance (MDR) efflux pumps driven by H(+) transport in Escherichia coli. While tolC expression is under the regulation of the EvgA-Gad acid resistance regulon, the role of TolC in growth at low pH and extreme-acid survival is unknown. METHODS AND PRINCIPAL FINDINGS: TolC was required for extreme-acid survival (pH 2) of strain W3110 grown aerobically to stationary phase. A tolC deletion decreased extreme-acid survival (acid resistance) of aerated pH 7.0-grown cells by 10(5)-fold and of pH 5.5-grown cells by 10-fold. The requirement was specific for acid resistance since a tolC defect had no effect on aerobic survival in extreme base (pH 10). TolC was required for expression of glutamate decarboxylase (GadA, GadB), a key component of glutamate-dependent acid resistance (Gad). TolC was also required for maximal exponential growth of E. coli K-12 W3110, in LBK medium buffered at pH 4.5-6.0, but not at pH 6.5-8.5. The TolC growth requirement in moderate acid was independent of Gad. TolC-associated pump components EmrB and MdtB contributed to survival in extreme acid (pH 2), but were not required for growth at pH 5. A mutant lacking the known TolC-associated efflux pumps (acrB, acrD, emrB, emrY, macB, mdtC, mdtF, acrEF) showed no growth defect at acidic pH and a relatively small decrease in extreme-acid survival when pre-grown at pH 5.5. CONCLUSIONS: TolC and proton-driven MDR efflux pump components EmrB and MdtB contribute to E. coli survival in extreme acid and TolC is required for maximal growth rates below pH 6.5. The TolC enhancement of extreme-acid survival includes Gad induction, but TolC-dependent growth rates below pH 6.5 do not involve Gad. That MDR resistance can enhance growth and survival in acid is an important consideration for enteric organisms passing through the acidic stomach

The Somatic Genomic Landscape of Glioblastoma

We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer

Osmolytes contribute to pH homeostasis of Escherichia coli

Background: Cytoplasmic pH homeostasis in Escherichia coli includes numerous mechanisms involving pH-dependent catabolism and ion fluxes. An important contributor is transmembrane K + flux, but the actual basis of K + compensation for pH stress remains unclear. Osmoprotection could mediate the pH protection afforded by K + and other osmolytes. Methods and Principal Findings: The cytoplasmic pH of E. coli K-12 strains was measured by GFPmut3 fluorimetry. The wild-type strain Frag1 was exposed to rapid external acidification by HCl addition. Recovery of cytoplasmic pH was enhanced equally by supplementation with NaCl, KCl, proline, or sucrose. A triple mutant strain TK2420 defective for the Kdp, Trk and Kup K + uptake systems requires exogenous K + for steady-state pH homeostasis and for recovery from sudden acid shift. The K + requirement however was partly compensated by supplementation with NaCl, choline chloride, proline, or sucrose. Thus, the K + requirement was mediated in part by osmolarity, possibly by relieving osmotic stress which interacts with pH stress. The rapid addition of KCl to strain TK2420 suspended at external pH 5.6 caused a transient decrease in cytoplasmic pH, followed by slow recovery to an elevated steady-state pH. In the presence of 150 mM KCl, however, rapid addition of another 150 mM KCl caused a transient increase in cytoplasmic pH. These transient effects may arise from secondary K + fluxes occurring through other transport processes in the TK2420 strain. Conclusions: Diverse osmolytes including NaCl, KCl, proline, or sucrose contribute to cytoplasmic pH homeostasis in E. coli

Effect of various osmolytes on cytoplasmic pH homeostasis.

<p>Fluorescence intensity was converted to cytoplasmic pH as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010078#s4" target="_blank">Materials and Methods</a> with the addition of 30 mM sodium benzoate to collapse the ΔpH and addition of KOH to raise the pH for the second standard pH point. Each trace shown is a representative replicate of three biologically independent cultures; panels <b>A</b> and <b>B</b> show two replicate curves for each condition, indicating the range of minimal variability seen throughout our experiments. (<b>A</b>, <b>B</b>): <i>E. coli</i> K-12 strains Frag1 and TK2420 transformed with the GFPmut3b reporter plasmid (pMMB311) were resuspended in M63A medium (5 mM MES; pH 5.6) with different osmolytes. Each panel includes Frag1 (black) and TK2420 (dark brown) cultures in M63A medium that contains less than 10 mM of each K⁺ and Na⁺. The other conditions included TK2420 with an additional 300 mM KCl (red), TK2420 with an additional 300 mM NaCl (blue), and TK2420 with 150 mM choline chloride (violet). (<b>C</b>, <b>D</b>): Strains Frag1 and TK2420 transformed with the GFPmut3b reporter plasmid were resuspended in M63A and subjected to a rapid osmotic upshift with the addition of 150 mM KCl. Each panel includes a TK2420 (brown) culture in media that contain less than 10 mM of both K⁺ and Na⁺. The other conditions included (<b>C</b>) Frag1 at pH 6.9 (gray), Frag1 at pH 5.6 (black), and TK2420 at pH 6.9 (light brown); (<b>D</b>) all TK2420 at pH 5.6: 150 mM KCl (red), 150 mM NaCl (blue), 150 mM choline chloride (violet), and 300 mM proline (cyan). Addition of KOH is not shown.</p

Acid and Base Stress and Transcriptomic Responses in Bacillus subtilis▿†

Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived <15%. Cells grown at pH 9.0 survived 40 to 100% at pH 10, whereas cells grown at pH 7.0 survived <5%. Thus, growth in a moderate acid or base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K+/H+ antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids

Effect of various osmolytes on cytoplasmic pH recovery after a rapid pH shift.

<p>Each trace shown is a representative replicate of three biologically independent cultures. Fluorescence intensity was converted to cytoplasmic pH as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010078#s4" target="_blank">Materials and Methods</a> with the benzoate and KOH additions not shown. (<b>A</b>, <b>B</b>): <i>E. coli</i> strain MC4100AR Δ<i>tatABCDE</i> TorA-GFPmut3* was resuspended in M63A (5 mM HOMOPIPES; pH 7.5) and subjected to a pH shift to pH 5.5 with 8.5 mM HCl at 0.6 min (arrow). The media contained 100 mM NaCl (blue), 100 mM choline chloride (violet), 100 mM KCl (red), 200 mM sucrose (orange), 200 mM proline (cyan), or no added osmolyte (brown). (<b>C</b>, <b>D</b>): <i>E. coli</i> K-12 strains Frag1 and TK2420 transformed with the GFPmut3b reporter plasmid (pMMB311) were resuspended in M63A (5 mM MOPS, 5 mM MES; pH 7.0) with different osmolytes and subjected to an acid shift to pH 6.0 with approximately 10 mM HCl. Each panel includes Frag1 (black) and TK2420 (brown) cultures in M63A that contains less than 10 mM of both K⁺ and Na⁺. The other conditions included TK2420 with an additional 300 mM KCl (red line), TK2420 with an additional 300 mM NaCl (blue), and TK2420 with 300 mM proline (cyan).</p

Effect of aeration on the extreme acid survival of W3110, Δ<i>hybC</i>, Δ<i>hycE</i>, and Δ<i>hypF</i>.

<p>The white bars represent cultures grown with aeration to stationary phase in LBK buffered at pH 5 that were diluted 200-fold into LBK pH 2 and exposed for 2 h with aeration at 37°C. The hatched and black bars represent anaerobic cultures grown to stationary phase in LBK buffered at pH 5.5 that were diluted 200-fold into LBK pH 2.5 (hatched) or pH 2 (black) and exposed for 2 h without aeration at 37°C. Aerobic and anaerobic cultures were maintained as stated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010132#s4" target="_blank">Materials and Methods</a>. Error bars represent SEM, n = 5 or 6.</p